Progesterone is an ovarian steroid hormone essential for breast development. The progesterone receptor (PR) exists primarily in two co-expressed isoforms, PR-A and PR-B. Studies from knockout-mice have shown that PR-B is required for proliferative signaling during mammary gland alveologenesis. In contrast, PR-A is required for uterine development, where progesterone inhibits proliferation. Very little is understood regarding regulation of PR tissue- and isoform-specific transcription. How are isoform-specific mitogenic (PR-B in the breast) and inhibitory/differentiative (PR-A in the uterus) effects achieved? PR-A and PR-B are most often co-expressed in the same tissues, and cells that express only a single PR isoform are rare, except in breast cancer where the normal 1:1 ratio is frequently altered. Selective PR isoform inhibition (blocking pro-proliferative effects of PR-B while preserving the protective or anti-proliferative effects of PR-A) would represent significant progress in breast cancer prevention and/or treatment. PR is highly post-translationally modified. Phosphorylation, primarily on PR-B N-terminal serine residues, significantly alters receptor stability, localization, transcriptional activity and promoter selectvity. Our preliminary data suggest that the basis for PR-B-specific proliferative actions in breast cancer cells involves ck2-dependent selective phosphorylation of PR-B Ser81 via a unique protein interaction domain, the common docking (CD) domain, found exclusively in PR-B but not PR-A. Phosphorylation of PR-B Ser81 regulates a highly specific subset of proliferative and pro-survival genes, including selected PR-regulated genes known to modulate the mammary stem cell compartment, such as Wnt1 and STAT5A. Additionally, our data suggest that STAT5A may serve as a pioneer factor, an early genomic binding partner that recruits/directs phospho-Ser81-PR-B-specific gene regulation. The goal of this research proposal is to determine how proteins that interact with PR-B via the CD domain regulate direct phosphorylation of PR-B on Ser81, thereby dictating PR-B isoform-specific transcriptional events at genes important for breast cancer cell proliferation, pro-survival, and expansion of the stem cell compartment. Hypothesis: PR and STAT5 co-regulate a specific subset of phospho-PR-B target genes (through CD domain- dependent recruitment of MKP3 and ck2, followed by ck2-mediated phosphorylation of PR-B Ser81) that regulate breast cancer cell proliferation and pro-survival, in part via modulation of the mammary stem cell compartment; selected genes are regulated by phospho-PR-B in the absence of ligand. ck2-dependent activation of PR-B may accelerate mammary tumor development and/or drive early breast cancer progression. PUBLIC HEALTH RELEVANCE: The goal of this research proposal is to determine how proteins that interact with the progesterone receptor (PR) regulate its direct phosphorylation, thereby dictating PR isoform-specific transcriptional events at genes important for breast cancer cell proliferation, pro-survival, and expansion of the stem cell compartment. Understanding how PR isoform-specific regulation is achieved may allow us to modulate/inhibit the proliferative actions of PR in the breast, while preserving protective anti-proliferative activities in other tissues. These studies could open the way to new treatments that may prevent or reverse the development of early cancerous steroid receptor positive breast lesions and/or provide novel PR-based additions to the current repertoire of largely estrogen receptor-based endocrine therapies.